LAB Exam 1 Flashcards

1
Q

What are the different methods for testing glucose?

A
  • Oxidation Reduction
  • Condensation reaction
  • Enzymatic reactions (3 types)
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2
Q

What are the three glucose enzymatic reactions?

A
  • Hexokinase (reference method)
    – Glucose oxidase-colorimetric (Influenced by heparin)
    – Glucose oxidase- polargraph (Influenced by heparin)
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3
Q

-Type of glucose Redox reaction that is not often used.
-Copper sulfate + Glucose –> Copper II oxide + Glucose-Na
-(In alkaline conditions & heat)

A

Benedicts- semi quantitative

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4
Q

-Glucose redox reaction
-Copper sulfate + Glucose —–> Copper II oxide + Glucose-Na
-Copper II oxide + phosphomolybdate —-> molybdenum blue

A

Folin Wu

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5
Q

Folin Wu reaction:

protein precipitation is….

A

Tungstic acid

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6
Q

Glucose redox reaction that uses Zinc sulfate and barium hydroxide

A

Somogyi Nelson

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7
Q

-Glucose redox reaction
-Ferric cyanide + Glucose —-> Ferrous cyanide (red color)

A

Automated Hagedorn Jensen

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8
Q

Condensation Reaction:

___________ condenses w/ Glucose in presence of Glacial acetic (hot)
producing blue green color (630 nm)

A

Ortho Toluidine

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9
Q

The condensation reaction for glucose can react with other aldoses like….

A

galactose and mannose

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10
Q

What kind of reactions are the most commonly used for testing glucose?

A

Enzymatic Reactions

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11
Q

Enzymatic Reaction:

Glucose + ATP ! G-6-P + ADP
G-6-P + NADP+ —> NADPH + 6-phosphogluconase

A

Hexokinase (reference method)

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12
Q

Enzymatic Reaction:

-Glucose + O2 —-> gluconic acid + H2O2
-H2O2 + Chromogenic peroxidase —-> Color + H20

A

Glucose Oxidase-colorimetric method (influenced by heparin)

-what we use in lab

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13
Q

Enzymatic Reaction:

Glucose + O2 —-> gluconic acid + H2O2

A

Glucose Oxidase- Polarography (influenced by heparin)

-needs electric current

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14
Q

What are the four lipid tests?

A
  • TRIGLYCERIDES
  • CHOLESTEROL
  • HDL ASSAYS
  • PHOSPHOLIPIDS
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15
Q

Why is TG testing done?

A

To calculate LDL using [LDL= total cholesterol-HDL-VLDL]
“ Friedewald equation”
-Assessment for CHD risk

-Determine whether increased TG are depressing HDL

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16
Q

High TG increases the risk of…

A

pancreatitis & numerous other clinical manifestations

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17
Q

How is TG and HDL related?

A

inversely related

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18
Q

What are the assays done for TG?

A

✓Hantzsch condensation
✓Sulfuric acid reaction
✓Pyruvate kinase/lactate dehydrogenase
✓Glycerol phosphate dehydrogenase/Diaphorase

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19
Q

What is the most frequently used “chemical” reaction for TG?

A

HANTZSCH CONDENSATION

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20
Q

Hantzsch condensation involves quantification of ________ using multiple steps

❖Organic extraction of TG
❖Chemical hydrolysis of TG to glycerol & FFA
❖Oxidation of glycerol to formaldehyde

A

glycerol

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21
Q

formaldehyde condenses with NH4+-Ac & acetylacetone to
form product which is measured between _____ and _____nm.

A

400 and 485nm

product measured is 3,5-DIACETYL-1,4-DIHYDROTOLUDINE

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22
Q

What are the first three steps of the Sulfuric acid reaction?

A

extraction, acidic/alkaline hydrolysis, & oxidation of glycerol

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23
Q

Sulfuric acid reaction:

utilizes chromotropic acid & sulfuric acid to produce a colored product upon reaction with formaldehyde

A

Eegriwe’s reaction

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24
Q

TRIGLYCERIDES —> GLYCEROL + 3 FATTY ACIDS (LIPASE)

GLYCEROL + ATP —> GLYCEROL-3-PHOSPHATE + ADP (GLYCEROL KINASE)

ADP + PEP —-> ATP + PYRUVATE (PYRUVATE KINASE)

PYRUVATE + NADH + H+ —-> LACTATE + NAD+ (LACTATE DEHYDROGENASE)

A

PYRUVATE KINASE/LACTATE DEHYDROGENASE

-frequently used assay

-enzymatic method for quantifying glycerol is multistep and measures NADH consumption

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25
PYRUVATE KINASE/LACTATE DEHYDROGENASE: NADH is measured at _____ nm.
340
26
PYRUVATE KINASE/LACTATE DEHYDROGENASE: What are the advantages?
1) Rapid and specific 2) Eliminates the need for extraction and caustic solvents
27
PYRUVATE KINASE/LACTATE DEHYDROGENASE: What are the disadvantages?
1) Instability of enzymes and reagents in working solution. 2) Serum blank must be included to subtract nonspecific absorption of various serum components.
28
PYRUVATE KINASE/LACTATE DEHYDROGENASE: When is a serum blank needed? How are results calculated?
Needed when sample: Hemolysis, Icteric, lipemic conditions water blank results - sample blank results = true O.D.
29
enzymatic method which employs the use of the same first two steps as the NADH consumption method.
FORMAZAN (COLORIMETRIC)
30
GLYCEROL-3-PHOSPHATE + NAD+ ---> DHAP + NADH (GLYCEROL PHOSPHATE DEHYDROGENASE) NADH + OXIDIZED TETRAZOLIUM ----> REDUCED TETRAZOLIUM (DIAPHORASE)
FORMAZAN (COLORIMETRIC)
31
employs only lipase/protease step to produce glyceride glycerol
FLUORIMETRIC METHOD
32
GLYCEROL-3-PHOSPHATE + NAD+ ---> DHAP + NADH (GLYCEROL PHOSPHATE DEHYDROGENASE) NADH + RESAZURIN ----> RESORUFIN (DIAPHORASE)
FLUORIMETRIC METHOD
33
TG: Plasma or serum samples must be from fasting individuals that have not ingested ALCOHOL for greater than ____ hours
36
34
_______ TG values are 2 to 4% lower than serum because of dilution effect of drawing water from red blood cells by the anticoagulant.
Plasma -remove the RBC from the plasma immediately
35
What is the anticoagulant of choice for TG testing? What should not be used?
EDTA Do not use glycerin coated vacutainers & care should be taken to avoid hand and body creams containing glycerol
36
What may interfere with TG assays?
Hemolysis, icterus and lipemia
37
What is the sample storage for TG testing?
-samples should be analyzed ASAP -or freeze at -60 degrees Celsius
38
What are the cholesterol methods?
* LIEBERMAN BURCHARD * SALKOWSKI * CHOLESTEROL OXIDASE/PEROXIDASE
39
* Described in 1885 (modified in 1889) * Common method and still in use today
LIBERMAN BURCHARD
40
Cholesterol Extraction, * React with sulfuric acid & acetic anhydride [sequential oxidation of cholesterol] * yields a blue-green cholesta-hexene-sulfonic acid
LIBERMAN BURCHARD
41
LIBERMAN BURCHARD: FREE CHOLESTEROL=
TOTAL CHOLESTEROL - ESTERIFIED CHOLESTEROL
42
LIBERMAN BURCHARD: What are the disadvantages?
1) DIFFERENT ESTERIFIED CHOLESTEROLS YIELD INCREASED COLOR INTENSITIES OF THE CHROMAGENS. 2) INTERFERENCES INCLUDE BILIRUBIN & UNREACTED DIGOXIN
43
-Chemical reaction first described in 1872 * Sample is extracted with chloroform -Addition of sulfuric acid changes the solution from BLUISH RED to VIOLET RED
CHOLESTEROL: SALKOWSKI
44
Salkowski: addition of sulfuric acid changes the solution from __________ to _________.
bluish red, violet red
45
Ferric chloride in Sulfuric acid was added to the Cholesterol in Acetic acid
Zlatkis and Boyle (1953) automated procedure
46
Zlatkis and Boyle (1953) automated procedure: Ferric chloride in Sulfuric acid was added to the Cholesterol in Acetic acid to give...
a more intense and stable magenta color ‣ Modification is 7x more sensitive than the Burchard method
47
What is the most popular and accurate method for measuring cholesterol?
CHOLESTEROL OXIDASE / PEROXIDASE
48
CHOLESTEROL OXIDASE / PEROXIDASE: ESTERIFIED CHOLESTEROL --> CHOLESTEROL + FA (__________) CHOLESTEROL + O2 ---> CHOLEST-4-EN-3-ONE + PEROXIDE (__________) PEROXIDE + PHENOL + 4-AMINOPHENAZONE ---> QUINOEIMINE + H2O
CHOLESTEROL ESTERASE CHOLESTEROL OXIDASE
49
CHOLESTEROL OXIDASE / PEROXIDASE: advantages?
INCREASED SPECIFICITY & NO HARSH REAGENTS
50
CHOLESTEROL OXIDASE / PEROXIDASE: disadvantages?
PEROXIDASE REACTION IS INHIBITED BY BILIRUBIN & SURFACTANTS
51
What specimen type is used for cholesterol testing? anticoagulant?
Serum or plasma EDTA -Others tend to cause large water shifts from the RBC to the plasma
52
Cholesterol testing: Should it be a fasting sample?
-12 hour fasting sample suggested -Cholesterol NOT directly affected by diet, except in patients with very high TG
53
What is important during the cholesterol sample draw?
-Posture is important during the draw because there can be as much as a 10 to 15% decrease after patient switches from standing to reclined position - Prolonged tourniquet use increases lipids.
54
What is the sample storage for cholesterol?
* PERFORM ASSAYS AS SOON AS POSSIBLE * IF THERE IS A DELAY, SAMPLE MUST BE FROZEN AND STORED AT -60°C (VORTEX SAMPLE WELL UPON THAWING)
55
HDL cholesterol is quantified by what two methods?
* APOLIPOPROTEIN B PRECIPITATION * ELECTROPHORESIS
56
* Lipoproteins are precipitated using low-density apoB lipoproteins (except HDL) with ________ solutions
polyanion
57
HDL- APO B PRECIPITATION: agents include...
▪ HEPARIN-MANGANESE CHLORIDE ▪ DEXTRAN SULFATE- MAGNESIUM CHLORIDE ▪ SODIUM PHOSPHOTUGSTATE ▪ POLYETHYLENE GLYCOL ▪ VARIATION-DEXTRAN-SULFATE WITH IRON ATTACHED (PPT W/MAGNET)
58
HDL- APO B PRECIPITATION: What is the reference method?
HEPARIN-MANGANESE CHLORIDE
59
HDL- APO B PRECIPITATION: Good Specificity for HDL
DEXTRAN SULFATE- MAGNESIUM CHLORIDE
60
HDL- APO B PRECIPITATION: Problem w/ Reagent Stability
SODIUM PHOSPHOTUGSTATE
61
HDL- APO B PRECIPITATION: Problems w/ Accuracy
POLYETHYLENE GLYCOL
62
HDL- APO B PRECIPITATION: advantages?
SIMPLE, FAST, AND INEXPENSIVE
63
HDL- APO B PRECIPITATION: disadvantages?
TENDENCY TO UNDERESTIMATE HDL
64
What are the HDL-CHOLESTEROL: ELECTROPHORESIS methods?
❖ STARCH BLOCK & GEON-PEVIKON BLOCK ELECTROPHORESIS ❖ AGAROSE GEL ELECTROPHORESIS ❖ POLYACRYLAMIDE GEL ELECTROPHORESIS
65
* SEPARATION OF LIPOPROTEINS BASED ON SIZE & NET CHARGE ✓HDL MIGRATES THE FASTEST ✓USED FOR ISOLATING LARGE QUANTITIES OF HDL BUT RARELY USED CLINICALLY
STARCH BLOCK & GEON-PEVIKON BLOCK ELECTROPHORESIS
66
✓SAME PRINCIPLE AS BLOCK ELECTROPHORESIS, but WITH A DIFFERENT RESOLUTION MATRIX * AFTER SEPARATION, AGAROSE GEL OVERLAYED WITH ENZYME REAGENTS FOR DETECTING CHOLESTER. AGAROSE STRIPS ARE SCANNED USING DENSITOMETER * NOT USED FOR ROUTINE ANALYSIS BECAUSE OF LOW RESOLUTIONS
AGAROSE GEL ELECTROPHORESIS
67
❖DATA WITH THIS TECHNIQUE CORRELATES WELL WITH ULTRACENTRIFUGATION
POLYACRYLAMIDE GEL ELECTROPHORESIS
68
Specimen collection and handling for HDL cholesterol?
* Patient should fast for 12 hours, although in most cases fasting does not affect HDL cholesterol * All other conditions are the same as for total cholesterol
69
What are the two types of liver function tests?
1. Intravascular injection of dye (indocyanine green) that is processed *solely* through liver (bile). i. Serum clearance of dye monitored 2. Injection of drugs to monitor liver’s ability to metabolize drugs - Appearance of modified drugs in serum or urinary excretion
70
Intravascular injection of dye liver test: Normal is ____ % dye remaining after 45 minutes.
<5
71
Intravascular injection of dye liver test: Higher retention times are indicative of...
hepatocellular disease or obstructive liver disease.
72
Why is Bromsulphalein (BSP) no longer used for intravascular injections?
due to “ANAPHYLAXIS”
73
Analytes for liver function tests?
▪ Enzymes ▪ Bilirubin ▪ Cholesterol ▪ Bile acids ▪ Serum proteins ▪ Urea and ammonia
74
Analyte that is helpful in differentiating the cause of jaundice*
BILIRUBIN
75
- 1) increased unconjugated bilirubin due to hemolysis; 2) serum bilirubin may be slightly increased, but liver will excrete large amounts of bile. 3) Increased urobilinogen in urine and feces. Urinary bilirubin will be absent.
Pre hepatic cause of jaundice
76
intermediate pattern, 1) unconjugated & conjugated bilirubin increased. 2) Fecal concentration of urobilinogen decreased but 3) conjugated bilirubin increased in urine.
Hepatic cause of jaundice -Hepatocellular damage / Bile excretion defect
77
1) increased conjugated bilirubin; serum conjugated bilirubin increased because of decreased biliary excretion. 2) Urobilinogen is decreased but bilirubin can now be detected in urine.
Post hepatic cause of jaundice
78
BILIRUBIN METHODOLOGY, what are the different types?
▪ Evelyn-Mallory ▪ Jendrassik-Groff ▪ Bilirubin oxidase ▪ HPLC ▪ Jackson Bilirubinometer ▪ Urine Bilirubin ▪ Bilirubin in amniotic fluid
79
Van den Bergh first discovered that bilirubin in serum reacted with ________ reagents
diazo
80
Reactions conducted directly on serum were hence called “direct” bilirubin measurements (actually measures ____________ bilirubin and δ-bilirubin)
hydrophilic conjugated
81
1883: Ehrich: Bilirubin (Urine) + ____________ ----> color
Diazotized Sulfanilic acid
82
* Unconjugated bilirubin (associated with albumin) required an “accelerator” (ex. Methanol) to dissociate from protein and solubilize bilirubin. Bilirubin that requires solvent is known as...
“indirect” bilirubin
83
Bilirubin + diazotized sulfanilic acid ----> 2 molecules azobilirubin
BILIRUBIN METHODOLOGY: EVELYN-MALLORY
84
“Direct” reaction performed at pH 1.2-azobilirubin (red purple) absorbs at 560nm in absence of solvent
BILIRUBIN METHODOLOGY: EVELYN-MALLORY
85
EVELYN-MALLORY: Total measurement conducted in presence of...
Methanol, urea, or DMSO Total – “direct” = “indirect”
86
What are the interferences with EVELYN-MALLORY method?
hemoglobin
87
What is the modified test of Evelyn-Mallory? What is different?
JENDRASSIK-GROFF “Direct” reaction carried out at pH 6.5 (NaOH used to alkalinize reaction) yields more intense blue color at 600nm
88
JENDRASSIK-GROFF: Total measurement conducted in presence of...
sodium benzoate, caffeine, methanol, urea or DMSO.
89
JENDRASSIK-GROFF: Total, indirect....
Serum (plasma) + Sodium acetate with caffeine-sodium benzoate
90
Enzymatic method that uses bilirubin oxidase to catalyze oxidation of bilirubin to biliverdin
BILIRUBIN OXIDASE
91
What absorbance is Bilirubin oxidase testing measured at?
decreased absorbance monitored at 405-460 nm
92
BILIRUBIN OXIDASE: Total bilirubin measured upon addition of detergent (____ or _______) added to dissociate bilirubin
SDS or Na-cholate
93
-pH of reaction manipulated to monitor conjugated bilirubin. ▪ Relatively new method, with promise of improved specificity and high precision
BILIRUBIN OXIDASE
94
What does HPLC stand for? What is it used for?
High Pressure Liquid Chromatography developed to separate methyl esters of conjugated & unconjugated bilirubin.
95
HPLC: Column elute is measured at ____ nm
430
96
Only means for accurate quantitation of bilirubin fractions. Interfering compounds are removed.
HPLC
97
HPLC: α: β:
α: unconjugated β: monoglucuronide
98
HPLC: γ: δ:
γ: diglucuronide δ: irreversibly bound to albumin
99
Direct spectrophotometric measurement of bilirubin at 454 nm
JACKSON BILIRUBINOMETER
100
JACKSON BILIRUBINOMETER: What is the drawback?
HbO2 absorbs at 454 nm, but HbO2 also absorbs at 540 nm Bilirubin level obtained by subtracting HbO2 contribution.*
101
Good screening method for neonates to 3 mo.; do not have significant carotenoids
JACKSON BILIRUBINOMETER
102
JACKSON BILIRUBINOMETER: Interference?
carotenoids absorb in same region and falsely elevate bilirubin levels.
103
In urine the only concern is __________ bilirubin
"direct"
104
What are the two methods to measure urine bilirubin?
1. Dipstick- Ehrlich diazo reagent 2. Ictotest- uses p-nitrobenzene diazonium p-toluenesulfonate
105
What is the most sensitive test to measure urine bilirubin?
Ictotest (2-4 times more sensitive than dipstick)
106
Conjugated Bilirubin + diazonium salt ----> azobilirubin (brown)
Dipstick- Ehrlich diazo reagent (2,4-dichloroaniline diazonium salt)
107
Bilirubin in amniotic fluid is measured by ________ spectrophotometric technique
DIRECT
108
High levels of bilirubin in Amniotic Fluid can indicate...
hemolytic disease -Most often associated with Rh incompatibility
109
Specimen storage for bilirubin testing?
Samples are stable in dark for 2 days at RT, 4 days at 4 degree C and indefinitely at -20 °C.
110
Bilirubin testing: Specimen should be protected from direct light. ▪ ___________ and ________ may effect determinations.
Hemolysis, lipidemia
111
Why is serum preferred for bilirubin testing?
proteins in plasma are likely to interfere with Evelyn-Malloy procedure
112
Infections precipitate ____% of hyperosmolar hyperglycemic states (HHS)
40-60
113
Major differences between HHS and other metabolic crises associated with diabetes?
-HHS is typically in older pts -HHS is often precipitated by an infection -acidosis does not occur in HHS, but still present with severe hyperglycemia
114
symptoms of HHS and DKA?
HHS- extreme thirst, dry mouth, dehydration DKA- nausea vomiting
115
What is the diagnostic criteria for hyperosmolar hyperglycemic state?
-Hyperglycemia- plasma glucose of grater than 600 mg/dl -plasma osmolality greater than 320 mOsm/kg -absence of ketoacidosis -dehydration -altered mental status -takes days to weeks to develop
116
What is the mechanism behind the pseudohyponatremia seen in hyperglycemic hyperosmolar state?
In HHS, blood glucose can be elevated to such a degree that osmolality increases as well. When this occurs, water moves from the intracellular space into the extracellular space, diluting the sodium concentration. In actuality, the concentration of sodium has not changed.
117
How do you calculate corrected sodium concentrations in the context of significant hyperglycemia?
-Methods such as the ion-selective electrode or using a corrected sodium formula that takes into account the serum glucose level. Corr. Na+ = [(Glucose – 100 mg/dl) * 0.016] + Na+
118
HHS is associated with what type of diabetes?
type 2
119
What enzymes are generally tested to determine liver function?
-Alanine transaminase (ALT) -Aspartate aminotransferase (AST) -Alkaline phosphatase (ALP) -Bilirubin
120
Released into bloodstream from damaged hepatocytes.
Alanine transaminase (ALT) and Aspartate aminotransferase (AST
121
Possible indicator of liver disease although can be present from damage to other tissues, like bone or the intestines.
Alkaline phosphatase (ALP)
122
Waste product produced when RBCs are broken down. Normally removed by the liver from the body through bile excretion into the GI tract. Declining liver function cannot remove bilirubin effectively so hyperbilirubinemia can occur.
Bilirubin
123
What are the expected values for direct bilirubin in adults?
Adults and infants (over one month): 0 – 0.5 mg/dl
124
What are the tests done to diagnose diabetes?
A1C, Fasting plasma glucose, Oral glucose tolerance test, and random plasma glucose test
125
What is the A1C threshold for diabetes mellitus?
6.5% or higher
126
measures the average blood glucose of the past several months.
A1C
127
Diabetes diagnosis: Fasting Plasma Glucose (FPG) that is ____ mg/dl or higher. The patient must fast for a minimum of 8 hours.
126
128
Diabetes diagnosis: Oral Glucose Tolerance Test (OGTT) that is ____ mg/dl or higher. This tests how well the patient can process sugar. It is done by testing blood glucose 2 hours before and after drinking a specific sweet drink.
200
129
Diabetes diagnosis: Random (Casual) Glucose Test that is ___ mg/dl or higher. This is a blood glucose test at any time of the day.
200
130
What chronic diseases are related to diabetes?
Cardiovascular disease, stroke, chronic kidney disease, diabetic neuropathy, vision loss.
131
What are the normal levels for fasting blood triglycerides in adults.
44-148 mg/dl (0.50-1.67 mmol/L)^9 (Pointe Scientific manual)
132
Hepatitis B
Most common chronic viral infection 42nm DnA virus hepadna virus family Made from RNA template (reverse transcription)—-> prone to mutation
133
Mutant prevents
HbeAg Mutant resistance to Reverse transcription inhibitor
134
HbsAg is
Common tests for Hep B
135
AntiHbcAg is also common
For testing Hep B
136
Liver function tests identify
Liver disease without jaundice
137
Monitor serum clearance of what
Indocyanine which is solely processed through liver(bile) and normal is 5% dye remaining after 45 minutes High retention time indicates liver disease
138
In the past Bromsulphalein was used but
Not today because of anaphylaxis toxicity
139
Bilirubin in four types
Alpha bilirubin= unconjagated bilirubin Beta bilirubin= 1 gluconic acid monocojagated bilirubin Gamma bilirubin= 2 glucocnic acid binding Delta bilirubin= reversely bound irreversibly to bilirubin
140
Alkaline phosphatase hydrolyzes Highest activity in
Hydrolyzes monophosphate esters Liver bone intestine and kidneys Placental obstruction increase activity about 10 fold
141
Hepatocytes damage but no release of
Alkaline phosphatase
142
Y- glutamyltransferase
Microsomal enzyme induced by drugs or alcohol Catalysts transfer of glutamate from glutathione to peptide
143
5’ nucleotidase-
Microsomal enzyme ( not elevated by drugs or alcohol Activity is solely in obstructive disease similar with AP
144
5’ nucleotidase can be used to exclude
Bond disease and damage
145
Lactate dehydrogenase
Cytosolic enzymes lactate-pyruvate Released after cell damage Viral/ toxic hepatitis, extra hepatic biliary obstructio, acute hepatic necrosis, and cirrhosis
146
5 types of lactate dehydrogenase
LD1- heart LD2- renal disease LD3- lung LD4- Skelton muscle LD5-liver
147
AST
Aspartate aminotransfersse and ALT alanine transaminase Converts aspartate and alanine to oxaloacetate and pyruvate respectively Most useful for detecting liver damage AST and ALT are also in the heart
148
Three types of jaundice
Pre hepatic Hepatic Post hepatic
149
Pre hepatic jaundice
Increase unconjugated bilirubin due to hemolysis Liver will secrete large amount of bile Increased urobilinogen in urine and feces Urinary bilirubin is absent
150
Hepatic jaundice
Uncojugated and conjugated bilirubin increased. Fecal concentration of urobilinogen decreased Conjugated bilirubin increase in urine
151
Post hepatic jaundice
Increased conjugated bilirubin, serum congated bilirubin increased because of decrease in bilirary excretion aka blockage Urobilinogen is decreased but bilirubin is in urine